The long-term objective of this R21 project is to develop a novel therapeutic strategy for osteoarthritis (OA) through a basic understanding of the role of tyrosine kinases in cartilage homeostasis. Tyrosine kinases such as Src and FAK are known to be involved in mechanotransduction of joint tissues, and their pharmacological inhibition is reported to be beneficial to cartilage maintenance. However, available data suggest that Src activity varies depending on subcellular locations and it can be up- or down-regulated by mechanical loading. Furthermore, preliminary studies indicate that partial silencing of Pyk2, another tyrosine kinase whose structure is similar to FAK, abolishes TNF?-induced cytoplasmic, but not membrane-bound, Src activation. Since global knockout of Pyk2 alters bone remodeling, Pyk2-mediated responses in cartilage and subchondral bone may alter their interactions at a bone/cartilage interface and the progression of OA. The question addressed in this project is the role of a network of tyrosine kinases (Pyk2, Src and FAK) in cartilage maintenance. Our working hypothesis is that Src/FAK activation at distinct subcellular locations (e.g., cytoplasm and plasma membrane) acts as a mediator of load-driven chondro-protection and regeneration, and that Pyk2 interacts with Src/FAK for anabolic and catabolic regulation of cartilage matrix. To test our hypotheses, we propose two specific aims using normal and OA cartilage explants (ex vivo), and wild type and Pyk2 knockout mice. Aim 1: Determine tyrosine kinase signaling in cartilage/subchondral bone explants Aim 2: Evaluate the role of Pyk2 in OA knee joints In the proposed ex vivo cartilage explant system, mechanical loading will be applied by a custom-made loading apparatus with and without OA induction. To determine kinase activities at the subcellular level, we will employ fluorescence resonance energy transfer (FRET)-based biosensors that are located at specific subcellular locations. Ex vivo measurements will be compared with in vivo mechanical loading data. We expect that advancing our understanding of the regulatory mechanism of Pyk2/Src/FAK in cartilage homeostasis will clarify merits and demerits of mechanotransduction of cartilage, and contribute to identifying a molecular target for treatment of degenerative joint diseases such as OA.